Traumatic brain injury (TBI) accounts for a significant number of casualties sustained by our soldiers, leaving many of them with substantial motor disabilities and cognitive impairments. There are no pharmacological interventions for brain trauma, mainly because the molecular and cellular mechanisms comprising brain injury remain unclear. Our current understanding of the pathogenesis of TBI suggests a dynamic interplay at the cellular level between excitotoxicity, oxidative stress, inflammatory events, and mitochondrial dysfunction, which occurs over days, weeks and months. Compelling evidence suggests that mitochondrial damage plays a crucial and central role in determining the outcome, since mitochondria are involved in regulating oxidative stress, inflammatory response, and cell death. The proposed studies are aimed at determining the mechanisms of mitochondrial damage focusing on a novel role of a toxic sphingolipid, sphingosine, as a cause of mitochondrial dysfunction following brain trauma. Our preliminary and published studies suggest that TBI provokes continued up-regulation of mitochondrial sphingosine, which could impact a number of mitochondrial functions, leading to persisting mitochondrial dysfunction after the primary insult. The long-term goal of the proposed studies is to develop a neuroprotective strategy based on attenuating sphingosine-dependent brain impairment after TBI. The central hypothesis of our proposal is that TBI-induced accumulation of sphingosine in mitochondria results in mitochondrial dysfunction, leading to neural cell injury and secondary brain damage. We plan to test our hypothesis by pursuing 2 specific aims: 1) Determine the mechanisms of the neutral ceramidase-mediated mitochondrial sphingosine generation and brain injury; 2) Determine the mechanisms of the acid sphingomyelinase-dependent mitochondrial sphingosine accumulation and brain injury. Fingolimod, an FDA- approved drug for treatment of multiple sclerosis, and Reclast, an FDA-approved drug for treatment of osteoporosis, will be utilized to inhibit sphingosine generation, reducing brain damage and preserving the brain function after TBI. We will use a multi-disciplinary and integrative approach, combining in vitro and in vivo studies in a mouse model of TBI with modern pharmacological, biochemical and bioenergetics methodologies. A powerful, state-of-the-art methodology, tandem mass spectrometry, will be utilized for assessment of sphingolipid- producing enzyme activities and precise measurement of the sphingolipid profile. The proposed studies will help us decipher novel sphingosine-mediated mechanisms of TBI and will lead to the development of effective therapeutic strategies to protect the brain from secondary injury, improving brain function recovery for Veterans with TBI.